Screening Of Sporozoite CDNA Library Of Eimeria Necatrix And Prokaryotic Expression And Characterization Analysis Of SNF2 Protein

Eimeria necatrix is one of the most pathogenic species among 7 species of chicken coccidia.Acute small intestinal coccidiosis caused by E.necatrix mainly occurs in 8 to 18-week-old chicken,and cause great economic losses to the poultry industry.With enormous pecuniary losses,there is an urgent need to find feasible prevention and control measures.The life cycle of avian coccidia includes three stages:sporogony,schizogony and gametogamy.Some key regulatory factors in the growth and development process may be effective targets for the prevention and control of avian coccidiosis.In the previous research,the cDNA library of the E.necatrix sporozoite was constructed by the SMART technology.On this basis,this study further screened the library to obtain the functional genes of E.necatrix,and expression and immunogenicity of the E.necatrix SNF2 gene.It will lay the foundation for the study of the E.necatrix subunit vaccine.Firstly,E.necatrix sporozoite cDNA libraries were screened by immunology and plasmid library methods to obtain EST sequences.(1)Immunological method:in immune screening,the library was screened using mouse polyclonal antibody as probe.40 suspected positive clones were obtained by primary screening.190 positive clones were obtained by rescreening.The rescreening positive phage XTriplEx2 were transformed into plasmid pTriplEx2,and then send to company to sequence.The EST sequences of 26 coccidia genomes were obtained,and were classified to 5 contigs.(2)Plasmid library method:the ?TriplEx2 phage library was directly transformed into the pTripIEx2 library,and 150 clones were selected and sent to the company for sequencing.A total of 53 EST sequences were obtained,and were classified to 21 contigs.Comparison of EST obtained from two methods,5 sequences are identical.The bioinformatics analysis of 21 contigs showed that these ESTs encode the SNF2 family helicase and elongation factor G involved in the DNA replication process,the cleavage factor involved in the RNA splicing process,and regulatory proteins such as serine protease inhibitors.Cell cycle-regulated PUA domain proteins,mitochondrial inner membrane tim17 subunit,domain proteins such as serine/threonine protein phosphatases,adenylate cyclase-related proteins,and sporozoite invasion-related microtubule proteins mic-1,ribosomal protein,acrosomal membrane antigen,MA2(ma-2)mRNA,Etm033F10 hypothetical protein,Etm087F12 hypothetical protein,Etm032G09 hypothetical protein,and Etml28F02 hypothetical protein,respectively.Secondly,the E.necatrix SNF2 gene,which was selected from the acquired EST sequences,was studied.Three pairs of primers were designed based on the E.necatrix SNF2 in GenBank.The sequence of the full-length SNF2 gene of E.necatrix was obtained by RT-PCR using segmented amplification and re-splicing.The amino acid sequence and functional domain of the gene were analyzed.Then,the N-terminal sequence of the SNF2 gene was expressed interceptively in the prokaryotic cell.The size of the expressed fusion protein was about 36 kDa,and it mainly existed in the form of inclusion bodies.The polyclonal antibody was prepared using the expressed product.Soluble protein of sporozoite was detected by Western blot with this polyclonal antibody.The size of the native SNF2 protein was found to be about 120 kDa,which is in line with the predicted theoretical value.Finally,chickens vaccinated with rEnSNF2 at different doses(200,100,50 ?g/feathers),and challenged with oocysts after the booster immunity except negative group.After two immunization,the experimental groups except the non-immunized group were all infection of E.necatrix sporulated oocysts and was used to evaluate the immune protective effect of recombinant proteins with the survival rate,oocyst reduction rate,lesion score,and average weight gain.The results showed that rEnSNF2 immuned with 200 ?g immunization per bird groups had the best protective effect.Compared with the negative group,the rEnSNF2 immuned groups could reduce lesion scores and oocyst yield,improve the survival rate,and induce specificity antibodies.